Chromosomal analysis is a technique used for screening or the diagnosis of genetic diseases, in an assay of the mutagenicity of physical or chemical factors, and other various cytogenetical purposes in medical, biological, agricultural or other fields. Chromosomes are obtained usually from mitotic cells using colhitin or its derivative, colcemid, thereby inhibiting the assembly of tubulin to form mitotic spindles during mitotic process. Therefore it requires cells to pass through mitosis. However, it is well known by those skilled in the art, that it is often difficult to obtain chromosomes in the case in which cells are not proliferate well.
Furthermore, many phenomena occur during interphase; for example, chromosomal cleavage by irradiation and subsequent reunion occur through the interphase nuclei, resulting in chromosomal damage. Thus, it has been required to establish a technique that allows one to obtain chromosomes from interphase cells.
Muller and Streffer (Muller et al. (1991) Int. J. Radiat. Biol. 59, 863-873) published a comprehensive review of biological indicators of radiation damage, explaining current techniques of biological dosimetry for radiation dose assessment. After exposure to high doses of radiation, sufficient numbers of mitotic cells are not available for dose assessment by the routine metaphase spread chromosome aberration analysis. The premature chromosome condensation (PCC) assay, performed on an exposed individual's blood lymphocytes, is viewed as a rapid biodosimetry method of clinical significance (Pantelias et al. (1985) Mutat. Res. 149, 67-72; Blakely et al. (1995) Stem Cells 13, 223-230; and Prasanna et al. (1997) Health Phys. 72, 594-600.
Currently, physical damage to chromosomes can be analyzed by observation of chromosomes after preparation of a metaphase spread. Chromosomes are visualized in mitotic cells following a short-term cell culture in which cells are stimulated into proliferation by a mitogen and then subjected to cell cycle arrest with colchicine or colcemid. The chromosomes are observed under a microscope after being treated either by staining or by hybridizing with a fluorescent probe. This technique depends upon the successful stimulation of the cells to proliferate and requires 48 hours or more of cell culture to obtain useful yields. The technique is labor intensive and requires experience in cytogenetic techniques to practice. The analysis is further complicated by cell killing and cell cycle delay induced by the treatment. In addition, the low yield of condensed chromosomes frequently requires large numbers of metaphase spreads to obtain statistically significant data.
Another method of analyzing physical damage to chromosomes involves inducing the premature chromosome condensation (PCC) in the cells and preparing a chromosome spread. Historically, premature chromosome condensation was accomplished by fusing the cells of interest with mitotic cells. This resulted in the condensation of the chromosomes in the test cells into chromatid-like structures. Although this technique does produce premature chromosome condensation, there are several difficulties associated with its practice. The technique requires a constant supply of mitotic cells to be fused with the test cells. The culture and maintenance of the mitotic cells adds considerably to the expense of the method. Additionally, cell fusion techniques (for example, PEG mediated fusion) are inefficient and produce low and variable yields of fused cells. This results in a low and variable yield of premature chromosome condensation in the test cells (Pantelias et al. (1983) Somatic Cell Genet. 9, 533-547).
The deficiencies of mitotic cell fusion to induce premature chromosome condensation are well known in the art and the search for alternative simple and rapid protocols has been a topic of ongoing research (Gotoh et al. (1996) Int. J. Radiat. Biol. 70, 517-520; Kanda et al. (1999) Int. J. Radiat. Biol. 75, 441-446; Durante et al. (1998) Int. J. Radiat. Biol. 74, 457-462; and Coco-Martin et al. (1997) Int. J. Radiat. Biol. 71, 265-273). Recently, premature chromosome condensation has been induced by stimulating cells with a mitogen and then culturing the cells in the presence of phosphatase inhibitors. Inhibitors of type 1 and 2A protein phosphatases have been used to induce PCC in proliferating cells (Gotoh et al. (1996) Int. J. Radiat. Biol. 70, 517-520; Kanda et al. (1999) Int. J. Radiat. Biol. 75, 441-446; Durante et al. (1998) Int. J. Radiat. Biol. 74, 457-462; and Coco-Martin et al. (1997) Int. J. Radiat. Biol. 71, 265-273).
The condensed chromosomes prepared by phosphatase inhibitor treatment were evaluated for biological dosimetry applications using chromosome aberration analysis in PCC spreads. Premature chromosome condensation was induced by okadaic acid (OA) (Gotoh et al. (1996) Int. J. Radiat. Biol. 70, 517-520; Kanda et al. (1999) Int. J. Radiat. Biol. 75, 441-446) or calyculin A (Durante et al. (1998) Int. J. Radiat. Biol. 74, 457-462) in mitogen stimulated cells and obtained 48 hours after mitogen-stimulation. Durante et al. (Durante et al. (1998) Int. J. Radiat. Biol. 74, 457-462) demonstrated that simultaneous measurement of chromosome aberrations in G1 and M phases is possible by using whole-chromosome probe fluorescence in situ hybridization (FISH) technique following exposure to 200-kVp x-rays. It has also been shown that incubation of actively dividing tumor cell lines in a cell culture medium containing OA or calyculin A results in PCC induction (Coco-Martin et al. (1997) Int. J. Radiat. Biol. 71, 265-273). Using whole-chromosome-specific probes, chemically induced PCC spreads containing radiation-induced chromosome aberrations are readily identified as cells with more than 2 chromosome spots. A difference in radiosensitivity was demonstrated between radiosensitive (SCC61) and radioresistant (A549) cell lines (Coco-Martin et al. (1997) Int. J. Radiat. Biol. 71, 265-273).
Although the use of phosphatase inhibitors produces premature chromosome condensation in stimulated or proliferating cells, presently available methods still require an incubation period in order to produce sufficiently high yields of premature chromosome condensation to be useful for chromosome aberration analysis. The current invention addresses the shortcomings in inducing PCC and in the treatment of certain disorders.